Cellular expression of epigenetic markers and oxidative stress in periodontitis lesions of smokers and non‐smokers

Abstract Objective To evaluate differences in the cellular expression of epigenetic markers and oxidative stress in periodontitis lesions between current smokers and non‐smokers. Background Tobacco smoking is recognized as one of the major risk factors for periodontitis. However, the mechanisms by which smoking affects the progression of the disease remain to be determined. Methods Twenty‐five current smokers and 21 non‐smokers with generalized severe periodontitis were included. From each patient, one soft tissue biopsy from a periodontitis site was harvested and prepared for histological analysis. The infiltrated connective tissue (ICT) was selected as the region of interest to assess the cellular expression of epigenetic markers and reactive oxygen/nitrogen species (RONS) by immunohistochemistry. Results Although the ICT of smokers and non‐smokers did not differ in size or in the expression of markers for DNA damage or oxidative stress, current smokers presented with significantly lower area proportions and densities of cells positive for the epigenetic markers DNMT1 and AcH3. In addition, periodontitis lesions in current smokers presented with a diminished antimicrobial activity, as indicated by significantly lower densities and area proportions of NOX2‐ and iNOS‐positive cells. Conclusions Components of the host response and epigenetic mechanisms in periodontitis lesions in smokers are downregulated as opposed to lesions of non‐smokers.

risk factors for periodontitis, [6][7][8][9] the mechanisms by which smoking affects the progression of periodontitis remain to be determined.
Chromatin arrangement is one of the main features of epigenetics and regulates gene expression without altering the DNA sequence. 10 DNA methyltransferases (DNMTs) are a family of enzymes that add a methyl group to cytosine-phosphate-guanine (CpG) islands on the DNA structure. This process is known as DNA methylation and induces the inactivation of DNA transcription, leading to gene silencing. Other epigenetic mechanisms involve the regulation of histones, that is, the structural proteins around which the DNA helix coils within the nucleosome. Acetyl groups on the histone tail may be removed or added by histone acetyltransferases (HATs) and histone deacetylases (HDACs).

F I G U R E 1
Histological sections stained with hematoxylin and eosin from periodontitis sites in a nonsmoker and a current smoker. Infiltrated connective tissue (ICT) outlined in black. Magnification ×20 Studies have shown that chemical substances in tobacco products generate genotoxic effects on the host immune response. 16,17 Thus, diminished chemotaxis and phagocytosis have been identified in neutrophils together with enhanced levels of markers for oxidative stress and production of reactive oxygen species (ROS). 18,19 NADPH-oxidases (NOX) and nitric oxide synthases (NOS) are two families of enzymes produced by neutrophils and macrophages, and contribute to the formation of superoxide 20  In a study on human biopsy material representing sites with severe periodontitis in current smokers and non-smokers, densities of vascular units and inflammatory cells were assessed. 23 While no differences in phenotype markers of inflammatory cells were detected between the two groups of samples, periodontitis lesions in smokers presented with fewer but larger vessels than lesions in non-smokers. The aim of the present study was to further analyze the histological material presented by Schmidt et al. 23 with a specific focus on differences in DNA methylation status and histone modification levels, and cellular expression of oxidative stress and reactive oxygen/nitrogen species between current smokers and non-smokers.

| Immunohistochemistry
After dehydration and embedding in paraffin, the biopsies were  Table 1. In brief, DNA methylation levels were identified by the DNMT1 and TET2 markers, while the histone acetylation levels were measured using the AcH3, AcH4, HDAC1, and HDAC2 markers. The γ-H2AX marker was used to identify DNAdamaged sites caused by double-stranded breaks, while the 8-OHdG marker was used to estimate the DNA damage caused by oxidative stress. The cellular expression of RONS (iNOS, NOX2) was also measured. Counterstaining was performed with hematoxylin. Finally, the sections were mounted and cover-slipped.

| Histological analysis
who was blinded to the origin of the samples. The infiltrated connective tissue (ICT) was depicted and outlined with a mouse cursor as the region of interest (ROI). The smart segmentation tool of the IPP software was used to identify each cell marker, using a differential method analysis of color, intensity, morphology, and size.
Thus, the total area occupied by positive cells was assessed for each marker and its percentage area relative to the total area of the ICT was calculated. In addition, the average cell size for each cell marker category was assessed. The number of positive cells in the ICT was computed using the data from the ROI total area, the average cell size, and the total area occupied by the positive cells. Cell numbers were expressed as total number and density of cells (number of cells/mm 2 ) within the ROI.

| Data analysis
The power calculation was based on assessments of cell phenotypes as reported previously. 23  Mean values and standard deviations were calculated for each variable, using the patient as the experimental unit. Differences between the two groups (current smokers and non-smokers) were analyzed using the Mann-Whitney U-test for independent variables.
The null hypothesis was rejected at p < .05.

| RE SULTS
Micrographs of periodontitis sites obtained from a current smoker and a non-smoker are presented in Figure 1 and a panel of micrographs representing each cellular marker in both groups is depicted in Figure 2. who reported that specimens representing gingivitis and healthy gingival tissue in smokers presented with higher levels of iNOS than those found in corresponding tissue samples from never-smokers. It should be realized, however, that the study by Özdemir et al. 31 did not include periodontitis lesions and did not assess cellular densities.
The finding in the present study on lower densities of NOX2positive cells in periodontitis lesions of current smokers indicates a disturbance in neutrophil functions, including the formation of neutrophil extracellular traps (NETs) and the elimination of pathogens through the "respiratory burst" process. 32,33 As NOX2 is part of the NADPH-oxidase family, studies showing associations between genetic polymorphisms of NADPH-oxidase and the condition "aggressive periodontitis" of the former classification are of interest (e.g., Nibali et al. 34 ).
Papapanou et al. 35 analyzed soft tissue biopsies obtained from patients affected by chronic or aggressive periodontitis. It was reported that a higher expression of NAD + , a precursor of NADPH, was noted in aggressive periodontitis sites than in chronic periodontitis sites.
Taken together, in addition to previous findings of fewer but wider vessels in periodontitis lesions of current smokers than in nonsmokers, 23 the results of the present study showed that important components of the host response and epigenetic mechanisms in periodontitis lesions in smokers are downregulated as opposed to lesions of non-smokers. It is important to note, however, that differential gene expression analysis was not conducted in the present study.
Furthermore, limitations concerning the interpretation of results obtained from the analysis of cellular markers in histological specimens should always be considered. In fact, the cellular expression of various markers in histological preparations does not necessarily reveal an ongoing process or molecular production. Nevertheless, since tissue-specific variability plays an important role in epigenetic mechanisms, a strength of the present study is the analysis of the entire ICT in well-preserved specimens. Lastly, although the power calculation was made for the preceding study on phenotype markers, the present sample size was sufficient to detect differences in selected markers between current smokers and non-smokers.
In conclusion, while a clear linkage between epigenetics and inflammation remains to be established, the results of the present study point to an association between epigenetic modifications and severe periodontitis depending on whether or not the patients smoke tobacco products. These findings should be considered in risk assessments and classification of periodontitis patients. Clinicians need to be aware that smokers require specific attention in treatment planning and maintenance.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.